The present invention relates generally to antimicrobial agents, and particularly to cationic amphiphilic polyproline helices as antimicrobial agents.
The emergence of drug resistant bacteria is a growing challenge to anti-infective therapy. Pathogens such as methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA), and resistant Pseudomonas aeruginosa and Acinetobacter baumanni have undermined most, and sometimes all, clinically usable antibacterial drugs. As new bacterial strains continue to evolve, the concomitant discovery of novel antibacterial agents has drastically lagged behind. Furthermore pathogens such as Bacillus anthracis and Brucella, classified as bioterrorism agents, pose an ongoing threat to security. The generation of new antibacterial agents with novel mechanisms of action, therefore, is of high importance.
Another challenge in the development of effective antibacterial agents arises from bacterial pathogens that have evolved to inhabit mammalian cells, such as phagocytic macrophages. Within these intracellular safe havens the bacteria reproduce and form a repository, causing recurrent infections. Such intracellular pathogenic bacteria include Mycobacterium tuberculosis, Salmonella, Listeria, Legionella and Brucella. When localized within mammalian cells, bacteria are able to evade the host immune response as well as a number of antibiotic drugs. A significant subset of antibiotics, such as p-lactams and aminoglycosides, are unable to achieve therapeutic concentrations within the host cell due to poor cell permeability and/or efflux transporters. These difficulties have spurred efforts to target intracellular pathogens using delivery vehicles such as liposomes and nanoparticles containing antibiotics.
Antimicrobial peptides (AMPs) are evolutionarily conserved molecules produced widely by plants and animals to defend against microbes. The majority of AMPs exert their antibiotic effect by targeting the microbial cell membrane resulting in cell lysis. This mechanism of action is a major drawback for clinical use of AMPs due to host cell toxicity. However, a small class of non-membrane lytic AMPs have been identified that have a high proline content and an overall cationic charge due to high levels of arginine, such as bactenectin, PR-39, apidaecin, pyrrhocoricin and drosocin. These proline-rich AMPs (P-AMPs) predominantly target Gram negative bacteria, while having minimal effect on Gram positive strains. In some cases the intracellular targets of P-AMPs have been identified such as the heat shock protein DnaK. While P-AMPs are less toxic than membrane-lytic AMPs, as seen in their lack of hemolytic activity and safety in animal models, the majority do not enter mammalian cells, with a few exceptions.
The plasma membrane of cells presents a formidable barrier to the passage of a number of therapeutic agents and probes. The passive uptake of genes, polypeptides and particles into cells is prohibitive due to size constraints, although smaller oligonucleotides and peptides are also too hydrophilic to adequately cross the hydrophobic bilayer. (Dokka, S. et al. (2000) Advanced Drug Delivery Reviews 44, 35-49; Juliano, R. (2007) Biochemical Society Transactions 35, 41-3.) The cell impermeable nature of these biopolymers and nanostructures serves to underscore the need to develop efficient strategies for their cellular uptake. A key feature to novel approaches is an understanding of the mechanism of cell penetration, so that better agents may be designed.
A number of different approaches have been taken to accomplish the delivery of therapeutic agents into cells. One recent approach has been the use of cell penetrating peptides (CPPs) that are rich in basic amino acids. (Fischer, R. et al. (2005) Chem. BioChem. 6, 2126-42; Futaki, S. (2005) Advanced Drug Delivery Reviews 57, 547-58; Vives, E. (2005) Journal of Controlled Release 109, 77-85.) CPPs have many advantageous features: generally low toxicity, high efficiency toward a variety of different cell lines, as well as the delivery of diverse cargo to intracellular targets reaching from proteins and oligonucleotides to magnetic nanoparticles. Two of the most well studied CPPs, derived from transcription factors, include the cationic domain of HIV-Tat (Tatp, GRKKRRQRRR)35 and the penetratin peptide from the Antennapedia homodomain. (Derossi, D. et al. (1994) Journal of Biological Chemistry 269, 10444-50; Derossi, D. et al. (1996) The Journal of Biological Chemistry 271, 18188-93; Prochiantz, A. (1996) Current Opinion in Neurobiology 6, 629-34.) Other short peptides used for membrane translocation include: cationic nuclear localization signal sequences, polylysine or polyarginine peptides, short peptides of alternating Arg and hydrophobic residues, peptides derived from the y-zein protein and other proline-rich sequences, and cationic moieties linked to scaffolds, such as peptoids, β-amino acid peptides, oligocarbamates, loligomers, dendrimers and biphenyls.
As can be seen, there is a need for effective antimicrobial agents, particularly those that can enter mammalian cells. While natural and synthetic peptides show promise as antimicrobial agents, it would desirable to have a peptide based antimicrobial agent that can enter mammalian cells by a number of different mechanisms, is toxic to microbes while sparing the host mammalian cell.